DNA replication:

Copying genetic information for transmission to the next generation Occurs in S phase of cell cycle Process of DNA duplicating itself Begins with the unwinding of the double helix to expose the bases in each strand of DNA Each unpaired nucleotide will attract a complementary nucleotide from the medium will form base pairing via hydrogen bonding.

Enzymes link the aligned nucleotides by phosphodiester bonds to form a continuous strand.1

DNA replication: First question asked was whether duplication was semiconservative or conservative Meselson and Stahl expt Semiconservative one strand from parent in each new strand

Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material.5

Meselson-Stahl experiments confirm semiconservative replication

Experiment allowed differentiation of parental and newly formed DNA. Bacteria were grown in media containing either normal isotope of nitrogen (14N) or the heavy isotope (15N). DNA banded after equilibrium density gradient centrifugation at a position which matched the density of the DNA: heavy DNA was at a higher density than normal DNA.Fig. 6.16 7

Meselson-Stahl experiments confirm semiconservative replication

When bacteria grown in 15N were transferred to normal 14N containing medium, the newly synthesized DNA strand had the 14N while the parental strand had 15N.

They checked the composition of the resulting DNA molecules by density gradient centrifugation, found an intermediate band, indicating a hybrid molecule containing both 14N and 15N DNA.8

15N

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The mechanism of DNA replication

Tightly controlled process, occurs at specific times during the cell cycle.

Requires: a set of proteins and enzymes, and requires energy in the form of ATP.

Two basic steps:

Initiation Elongation.

Two basic components:

template primer.

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The mechanism of DNA replication (prokaryotic)

DNA polymerase the enzyme that extends the primer; Pol III produces new stands of complementary DNA Pol I fills in gaps between newly synthesized Okazaki segments

additional enzymes/proteins i) DNA helicase

Origins of Replication Replication proceeds in both directions (bidirectionally) from a single origin of replication on the prokaryotic circular chromosome Replication proceeds in both directions (bidirectionally) from hundreds or thousands of origins of replication on each of the linear eukaryotic chromosomes.14

Origins of Replication Bacteria have 1 origin of replication per one chromosome They only have one chromosome = 1 origin!

What Really Happens.

Replication Termination The ends of chromosomes (telomeres) cannot be replicated on the lagging strand because there is no primer available. Telomerases enzymes that contain RNA primers which extend the ends of chromosomes (not normally expressed in significant levels) Telomeres form a sort of single stranded cap around the chromosome ends to protect them from being degraded

chromosome ends are progressively shortened with each round of replication. old cells with shortened telomeres undergo apoptosis Protective for normal cells Kill the old and possibly mutated

Telomerase is over expressed in cancer cells Hypothesis is that cancer cells do not undergo apoptosis because their telomeres do not shorten over time. No death signal22

Fig. 11.14 The problem of replicating completely a linear chromosome in eukaryotes

Replicating the Ends of Chromosomes

telomerase adds an RNA primer complementary to telomere sequences chromosomal replication proceeds by adding to the 3 end of the primer

Fills the gap left behind by replication Telomerase enzyme can also add DNA basepairs to the TEMPLATE DNA complementary to the RNA primer basepairs Using an RNA template to make DNA, telomerase functions as a reverse transcriptase called TERT (telomerase reverse transcriptase). This goes against the Central Dogma. Evolutionarily thought to be derived from a Retrovirus

The bidirectional replication of a circular chromosome (Prokaryotic)

Assembling Newly Replicated DNA into Nucleosomes

When eukaryotic DNA is replicated, it complexes with histones. This requires synthesis of histone proteins and assembly of new nucleosomes.

Transcription of histone genes is initiated near the end of G1 phase, and translation of histone proteins occurs throughout S phase. Assembly of newly replicated DNA into nucleosomes is shown in Figure 11.16.28